Author

Date of Award

Degree Type

Degree Name

Department

First Advisor

Bumsoo Han

Committee Chair

Bumsoo Han

Committee Member 1

Sherry L. Voytik-Harbin

Committee Member 2

Thomas Siegmund

Committee Member 3

J. Craig Dutton

Committee Member 4

Xianfan Xu

Abstract

Hierarchical structural interactions between components of cell microenvironment, the extracellular matrix (ECM), cytoplasm, nucleus and fluid, are important phenomena that decide cell level physiological process and tissue engineering applications. One of those tissue engineering modalities is freezing of biomaterials, important in a wide variety of biomedical applications including cryopreservation and cryosurgeries. In order to design these applications, freezing-induced changes of the cells and tissues and corresponding biophysical mechanisms need to be well understood. Although the effects of freezing on cells in suspension have been extensively studied, the intracellular mechanics of cells embedded in the extracellular matrix (ECM) during freezing are not well understood. Since the cells embedded in the ECM are subjected to the mechanical transmission of freezing induced ECM deformation, it was hypothesized that the subsequent intracellular deformation depends on the cytoskeletal structure, configuration of cell-ECM adhesion and deformation at cell-ECM interface which are anticipated to determine the outcome of freezing. ^ To quantify spatiotemporal deformation in cells, a new method, `particle tracking deformetry' (PTD), was developed using fibronectin coated polystyrene beads, that were internalized by cells. Fibroblast-seeded dermal equivalents were used as a model tissue. Effects of particle size on the deformation measurement method were tested, and it was found that microbeads represent cell deformation to acceptable accuracy. The results showed complex spatiotemporal deformation patterns in the cells. Large deformation in the cells and detachments of cells from the ECM were observed. At the cellular scale, variable directionality of the deformation was found in contrast to the one-dimensional deformation pattern observed at the tissue scale, as found from earlier studies. Subsequently, cytoskeletal structure and cell-ECM adhesion configuration was varied to test the effect of poroelastic parameters of cell on intracellular deformation. It was observed that the extent of intracellular deformation was highly dependent on the cytoskeletal structures. Rupture of cells and sudden void formation was noted inside cell during the process. With a given cytoskeletal structure, the nucleus and cytoplasm regions showed starkly different deformation behavior. The freezing-induced change in cell, nucleus, and cytoplasm volume were quantified and was correlated to intracellular deformation. To further understand the mechanical transmission from ECM to cell, deformation on both domains was quantified using a newly developed method 'dual domain deformetry'. Beads of different sizes were used in two domains to quantify the individual deformation pattern. Deformations in the two domains were found to be significantly different in terms of magnitude, directionality and homogeneity. ^ The methods developed can quantify the spatiotemporal deformation in and outside cells and can be correlated to the freezing-induced change in the structure of cytosplasm and of the cell-ECM interface. The biophysical insight generated from this study has implications in tissue-type independent cryo-technology protocol development and general tissue engineering applications. As a broader application, this method may be used to compute deformation of cells in the ECM environment for physiological processes, namely cell migration, stem cell differentiation, vasculogenesis and cancer metastasis, which have relevance to quantify mechanotransduction.